High Pressure Liquid Chromatography and It's Application

detailed study about the study of high pressure liquid chromatography

1. Presented by
Megha Hiroji

2. What is HPLC?
It is the fastest growing analytical technique for analysis of
chemicals especially for drugs
Some synonyms of the technique are:
HIGH PRESSURE LIQUID CHROMATOGRAPHY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HIGH PRICE LIQUID CHROMATOGRAPHY
HIGH SPEED LIQUID CHROMATOGRAPHY
HIGH EFFICIENCY LIQUID CHROMATOGRAPHY

3. Introduction
HPLCwasreferredtoasHighpressureLiquid
Chromatographybecausehighpressureisusedto
increasetheefficiencyofseparationbutnowadays
thetermHighperformanceLiquidChromatography
ispreferredasitdescribesthecharacteristicsofthe
chromatography.

4. Principle
Theresolvingpowerofacolumnincreaseswith
increaseincolumnlength,asnumberoftheoretical
platesperunitlengthincreases,itmaysometimelead
tobroadeningofpeakduetoincreaseinlengthof
column.
Thenumberoftheoreticalplatescanbeincreasedby
decreasingtheparticlesizeofstationaryphase.
Astheparticlesizedecreases,theresistancetoflowof
mobilephaseincreases.Thiscanbeovercomeby
increasingtheflowratesbymeansofemploying
pressurepumpingsystems.

5. THEORIES OF HPLC
PLATE THEORY:
Accordingtothistheory,thechromatographiccolumn
consistsofcontinuous,narrow,horizontalplatesof
equalunitscalledas“theoreticalplates”.
Thetheorymeasuresthecolumnefficiency&the
factorswhichinfluencetheefficiencyare:
Numberoftheoreticalplates(N)
Heightequivalenttotheoreticalplates(HETP)
Resolution

6. Number of theoretical plates (N):
Efficiency of a column is expressed by the number of
theoretical plates. It can be determined by
N= Number of theoretical plates
r =Retention time (min or sec)
w=peak width at base (mm or cm)
If the number of theoretical plates is high, then the
column is said to be highly efficient.
If the number of theoretical plates is low, then the
column is said to be less efficient

7. Height equivalent theoretical
plates (HETP):
Itisthelengthofthecolumnnecessarytoobtainthe
equilibriumofthesoluteparticlesbetweenthetwo
phases&isgivenby
HETP=lengthofthecolumn/no.oftheoreticalplates
WhereL=lengthofthechromatographiccolumn
N=no.oftheoreticalplates

8. RATE THEORY
This theory explained the fact that the mobile phase flows
continuously & that the solute particles are constantly
being transported & partitioned in the column. It can be
explained by Van Deemeterequation:
Where , h=HETP
λ=measure of packing irregularities of stationary
phase
d
p= particle diameter of the stationary phase
γ = tortuosity
D
g =diffusivity of the solute in mobile phase
d
f=film thickness

9. TYPES OF HPLC TECHNIQUES
A. Based on modes of chromatography
1.Normal phase mode
2.Reverse phase mode
B. Based on principle of chromatography
1.Adsorption chromatography
2.Ion exchange chromatography
3.Size exclusion/gel permeation chromatography
4.Chiral chromatography
C. Based on elution techniques
1.Isocratic separation
2.Gradient separation

10. 1.Normal phase chromatography
Stationary phase Polar and Mobile phase is
non polar
Stationary phase : silica, Alumina, -NH
2, -
CN, -Diol.
Mobile phase: Heptane, hexane, cylohexane,
CHCI
3, dioxane.
Application: Separation of non-polar to
medium polar substances

11. 2.REVERSE PHASE MODE
CHROMATOGRAPHY
Stationary phase non Polar and Mobile phase is
polar
Stationary phase: n-octadecyl(PR-18),n-octyl (RP-
8), ethyl(RP-2).
Mobile phase: Methanol or Acetonitrile/ water or
buffer

12. STATIONARYPHASES
Polar (“Normal” Phase):
Silica, alumina
Non-Polar (“Reversed Phase”)
Cyano, amino

13. The Mobile Phase in HPLC
1)Must be compatible with the instrument
2)Compatible with the stationary phase
3)Readily available
4)Of adequate purity
5)Not too compressible (causes pump/flow problems)

14. Eluotropic series for some
common solvents

15. INSTUMENTATION OF HPLC

17. Mobile phase delivery system
Twoormoreglassorstainlesssteelreservoirs,eachof
whichcontains500mlormoreofsolvent.
Themobilephasemustbedeliveredtothecolumnat
highpressureoverwiderangeofflowrates.
Thereservoirareoftenequippedwithameansof
removingdissolvedgassesusuallyoxygenand
nitrogenthatinterferebyformingbubblesinthe
columnsanddetectorsystem.

18. Solvent treatment system
Solvents of purified form are commercially available
for HPLC.
All solvents must be degassed before introduction into
pump as it can alter the resolution of column and
interfere in the monitoring.

19. DEGASSING SYSTEM
By warming the solvent
By stirring vigorously with magnetic stirrer
By subject to vacuum filtration
By ultrasonication
By bubbling the He gas
The solvents are delivered at
High pressure into the column
By means of pump.

20. PUMPS
1.To generate pressure.
2.To maintain constant flow rate.
3.To maintain constant flow reproducibility.
4.Resistant to the solvent.
Pumps are thus categorized into
1.Mechanical pumps
Displacement pumps
Reciprocating pumps
2.Pneumatic pumps

21. MECHANICAL PUMPS
1.DISPLACEMENT PUMPS
Worksontheprincipleofpositivesolvent
displacementbyapistonmechanicallydrivenata
constantrate.
Thepistonisdrivenbyascrewthroughagearboxbya
digitalsteppingmotor.
Therateofflowofmobilephaseiscontrolledby
changingthevoltageofthemotor.
Thesolventchamberhasonlyafinitecapacityand
mustberefilled.
Byemployingthesepumpshighpressurecanbe
achievedwithoutpulses.

22. ADVANTAGES
Flow is pulse free.
Provide high pressure up to 200 –475 atm.
Independent of column back pressure and viscosity of
solvent.
Simple operation.
DISADVENTAGE
Limited solvent capacity
Gradient elution is not easy.

23. 2. RECIPROCATING PUMPS
They are relatively inexpensive and permit a wide
range of flow rates.
It consist of a motor driven piston moves rapidly back
forth

24. ADVANTAGES
Provide wide range of flow rate
Easy to operate
Inexpensive
DISADVENTAGE
These pumps may produce pulsating flow of mobile
phase.

25. 3. Pneumatic pumps
In this the mobile phase is contained in a collapsible
container housed in a vessel that can be pressurized
gas.
Advantages:
Flow is pulse free.
Equipment is cheap.
Disadvantages:
Limited capacity
Gradient elution is not possible.

26. PRE-COLUMNS
A pre-column is packed with silica particles.
It is being fitted between pump & the injector valve
ensures that the mobile phase is fully saturated with
the silicate ions prior to the sample injection.
Thus its use reduces the adverse effects of low or high
pH mobile phases which in turn substantially extend
the life of the column. Their use is recommended in
ion-exchange chromatography using buffered aqueous
phase.

27. GUARD COLUMN
A short column placed between the sample injector and
the inlet of the main ("analytical") column
The guard column is packed with the same kind of
packing as the main analytical column, and
is intended to absorb or pick up impurities in the
sample or mobile phase that might damage the main
column & increase the life time of main
analytical column.

28. FLOW SPLITTER
When a differential type of detector is used the flow of
solvent is split just before it enters the sample injection
port
so that one portion directly goes to the reference side
of the detector & a portion to the analytical column
housed in a constant temperature chamber

29. SAMPLE INJECTOR PORT
a.Septum injector
b.Stop flow septum less injection
c.Rheodyneinjectors

30. Syringe is used to inject the sample through a self
sealing inert septum directly into the mobile phase.
Drawback: -leaching effectof the mobile phase with
the septum resulting in the formation of ghost peaks.
STOP FLOW SEPTUMLESS INJECTION.
Flow of mobile phase through the column is stopped
for a while.
Syringe is used to inject the sample.
Drawback: formation of ghost peak.

31. RHEODYNE INJECTORS
Sample is loaded at atmospheric pressure
into an external loop in the micro volume
sampling valve, and subsequently injected
into the mobile phase by suitable rotation
of the valve.

32. Analytical Column
Analytical column is most important part of the
instrument . Dimensions of column are
Column length: 5cm to 30cm
Column diameter: 2mm to 50mm
Particle size: 1μ to 20 μ
Particle nature: Spherical, uniform sized, porous
materials are used

34. Detectors
A detector is required to sense the presence and the
amount of sample component in the column
eluent.theout of the detector is an electrical signal
that is proportion to some property of the mobile
phase and or the solute.
UV visible detector
Flourimetric detector
Refractive index detector
Multiple detector

35. Recorder
The signals from a detector are recorded as deviation
from base line.
The peak position along the curve relative to the
starting point denotes the particular component.
Proper calibration, the height or area of the peak is a
measure of amount of component in a sample.

36. Columns
Types of columns
Guard column
Analytical column

37. Guard Column (Pre-column)
Placed between injector and analytical column
Same material as that of analytical column
Does not contributes to any separation
Eliminate particulate and impurities
Extend life time of analytical column
Protect the system component

38. Analytical Column
Most important part of HPLC technique which decides
efficiency of separation.
1.Radial Compression columns
2.Narrow-Bore Columns
3.Short , Fast Columns

39. Column Material
Stainless steel
Glass
Polyethylene
Polyether ether ketone (PEEK)

40. Column Dimensions
Column length
10-30 cm long
Internal diameter
5-10 mm

41. Column Preparation
Bottom portion of the column is packed with cotton wool
or glass wool or may contain asbestos pad, above which
adsorbent is packed.
Whatman filter paper disc can be used.
After packing the column with the adsorbent, a similar
paper disc is kept on the top so that the adsorbent layer is
not disturbed during introduction of sample

42. Packing Techniques
Dry packing
Wet / slurry packing

43. Column Packing Material
Micro porous supports:
3-10 µmin diameter
Composed of silica, alumina, ion exchange resin
Pellicular supports:
40 µmin diameter
Porous particles are coated onto an inert solid core such
as glass bed.
Bonded phases:
Stationary phase is chemically bonded onto inert
support.

44. Factors Affecting Column
Efficiency:
Dimensions of column
Particle size of adsorbent
Nature of solvent
Temp. of column
Pressure
Packing of column

45. Detectors
BASIC DETECTOR REQUIREMENTS
An ideal LC detector should have the following
properties
Low drift and noise level (trace analysis).
High sensitivity.
Fast response for high performance systems.
Wide linear dynamic range (quantization).
Low dead volume (minimal peak broadening &
remixing of the separated bands).
Insensitivity to changes in type of solvent, flow
rate, and temperature.
Operational simplicity and reliability.
Preferably non-destructive

46. Detectors
UV/Visible
Fixed wavelength
Variable wavelength
Refractive index
Fluorescence

47. ULTRAVIOLET VISIBLE DETECTOR
UV detectors are the most commonly used detector.
They measure the ability of a sample to absorb light.
This can be accomplished at one or several
wavelengths.
A variable wavelength UV detector, capable of
monitoring from 190 to 460-600 nm will be found
suitable for the detection of the majority of samples.

48. Mobile phase from the column is passed through a
flow cell held in the radiation beam of uv/ visible
spectrophotometer.
Selective in nature, detect only those solutes that
absorb uv/ visible radiation
e.g. alkenes, aromatic compounds and compound
having multiple bonds between C and O, N or S.
BASICALLY TWO TYPES OF ABSORBANCE
DETECTORS ARE AVAILABLE
fixed wavelength detector
variable wavelength detector

49. Fixed wavelength detector

50. HPLC detectors which do not allow changing the
wavelength of the radiation called fixed-wavelength
detectors.
Low-pressure mercury lamp emits very intense light at
253.7 nm.
By filtering out all other emitted wavelengths, utilize
only 254 nm line to provide stable, highly sensitive
detectors capable of measuring sub nanogram
quantities of any components which contains aromatic
ring.
The 254 nm was chosen since the most intense line of
mercury lamp is 254 nm, and most ofUV absorbing
compounds have some absorbance at 254 nm.

51. VARIABLE-WAVELENGTH
DETECTORS
Detectors which allow the selection of the operating
wavelength called variable wavelength detectors.

53. Sensitivity for any absorptive component by selecting
an appropriate wavelength individual sample
components have high absorptivity at different
wavelengths and thus, operation at a single wavelength
would reduce the system's sensitivity
Depending on the sophistication of the detector,
wavelength change is done manually or programmed
on a time basis into the memory of the system.

54. Reflective type of refractometer:
Measure change in % of reflected light at glass liquid
interface as the reflective index of liquid changes.
Based on the Fresnels law of reflection which states
“The amount of liquid reflected at a glass-liquid
interface varies with the angle of incidence and the
refractive index of the liquid”

55. working:
Two collimated beams from the projector (light source
& lens) illuminate the reference and sample cell.
Cells are formed of Teflon gasket, which is clamped
between the cell prism and a stainless steel reflecting
back plate.

57. As the light of beam is transmitted through the cell
interfaces, it passes through the liquid film and
impinges on the surface of the reflecting back plate.
Diffused, reflected light appears as two spots and
passes through the lens and detected by photo
detector.
The ratio of the reflected light to transmitted light is
function of refractive index of the two liquid, the
illumination of the cell back plate is direct measure of
the refractive index of the liquid in each chamber

58. FLOURIMETRIC DETECTORS
Very sensitive, but very selective.
It is possible to detect even a presence of a single
analyte molecule in the flow-cell.
Fluorescence occurs when compounds having specific
functional groups are excited by shorter wavelength
energy and emit higher wavelength radiation.

60. Fluorescence is often collected at right angle to
excitation beam.
Only one sixth of fluorescence is collected. If concave
mirror is placed around the sample cell abut 75 % of
the emission is collected.
With all sample cells, scattered radiation from the
excitation source is selectively removed with cut off or
band pass filters placed before photomultiplier tube.

61. APPLICATION OF HPLC
Quantitative analysis
Qualitative analysis
Checking the purity of the compounds
Isolation & identification of drugs or metabolites in
urine, plasma, and serum etc.
Stability studies
Purification of some compounds natural and synthetic
origin

62. Comparison
GAS
CROMATOGRAPHY
(GC)
DEFINITION:-
GC is a basically
separation techniques in
which a component of
vaporized sample are
separated and
fractionated as a
consequence of partition
b/w a mobile phase and a
stationary phase held in
column.
HIGH PERFORMANCE
LIQ.CHROMATOGRAP
HY(HPLC)
DEFINITION:-
HPLC is a process of
separation of mixture
containing two or more
component under high
pressure by passing the
sample through column
containing a stationary
phase by means of
pressurized flow of liq.
phase.

63. Classification:-
1.Gas solid
chromatography
2.Gas liquid
chromatography
PRINCIPLE:-
separation occur
on the basis of
partition
coefficient and
adsorption only
Classification:-
1.liq-liq chromatography or
partition HPLC
2.liq.-solid
chromatography or
adsorption
chromatography
3.Ion exchange HPLC.
4.Gel/size exclusive HPLC.
PRINCIPLE:-
separation occur on the
basis of partition
coefficient, adsorption,
ion exchange and size
exclusive

64. Ideal properties:-
It should be inert.
Suitable for sample and
detector analyzed.
Should be highly pure.
Should not cause risk
of explosion.
Ideal properties:-
Should be less viscous
than water.
Should be free from
oxygen and other
dissolve gas in mobile
phase.
Should be non –
inflammable in nature

65. Stationary phase
and mobile
phase.
Uncoated solid material
for GSC and coated with
a thin layer of liq. for
GLC.
-polyethylene siloxane
-polyethylene glycol
-diethyleneglycol
succinate
Stationary phase
and mobile
phase.
Octadecyldimethylsilyl
chloride is used as
stationary phase.
Mobile phase:-
Methanol
Acetonitrile
Chloroform
Ethanol
cyclohexane

67. COLUMN:-
length is long 120 to 150
cm in length and 4 mm
in diameter
1.packed column
2.capillary column
COLUMN
PACKING:-
Done using by Teflon,
glass beads, porous
polymers, adsorbent
such as zeolites,
alumina, silicate, etc
COLUMN:-
column size 10 times
smaller then GC
10 to 30 cm long
4 to 10 mm internal
diameter
1.Analytical column
2.Preparative column
COLUMN
PACKING:-
Done using by silica gel,
glassbeadsetc.

68. Pumps:-
No pump are used in
GC.
Pumps:-
Used to generate
pressure up to 5000 psi
to mobile phase to pass
through the column.
Reciprocating pump
Constant pressure
pump
Pneumatic amplifier
pump
Displacement pump

69. Detectors:-
Thermal conductivity
detector
Electron capture
detector
Flame ionization
detector
Argon capture detector
Nitrogen phosphorus
detector
Mass selective detector
Helium ionization
detector
Detectors:-
UV visible detector
Flourimetric detector
Refractive index detector
Multiple detector

70. Advantage:-
1.A strong separation
power.
2.Results obtained rapidly.
3.Applied for widely
divergence sample.
4.Simplicity and reliability
of operation of
equipment.
5.Relatively high precision.
6.Sensitivity detection is
very high.
7.The speed of analysis is
very fast.
8.Do not req. high skilled
person.
Advantage:-
1.fastest,simple,specific
and wide range of
sensitivity.
2.Analysis can be done in
dosage form and
biological fluids.
3.Efficiency is 5 to 10 times
more then other
methods.
4.Most widely used for
assay.
5.Easy of sample
preparation and sample
introduction.

71. DISADVANTAGE:-
Material has to be
volatized at 250
0
C
without decomposition
DISADVANTAGE:-
Very costly techniques.
Req. high skilled
person.

72. REFRENCES
Fundamentals of Analytical chemistry by Skoog,
West, Holler, Crouc
Pharmaceutical Analysis -Dr. A. V. Kasture.
Instrumental Analysis by G.R.Chatwal
Instrumental method of ofanalysis and technique
by, B.K. Sharma
Internet source